Clinical Science (2012) 122, 133142 (Printed in Great Britain) doi:10.

1042/CS20110233

Protein metabolism and gene expression

in skeletal muscle of critically ill
patients with sepsis

Maria KLAUDE , Maiko MORI , Inga TJADER

, Thomas GUSTAFSSON,

Jan WERNERMAN and Olav ROOYACKERS

Department of Anaesthesiology and Intensive Care, Karolinska University Hospital Huddinge, Karolinska Institutet,
S-14186 Stockholm, Sweden, Department for Clinical Science, Intervention and Technology and Clinical Physiology,
Karolinska University Hospital Huddinge, Karolinska Institutet, S-14186 Stockholm, Sweden, and Department of Laboratory
Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet, S-14186 Stockholm, Sweden

Muscle wasting negatively affects morbidity and mortality in critically ill patients. This progressive
wasting is accompanied by, in general, a normal muscle PS (protein synthesis) rate. In the
present study, we investigated whether muscle protein degradation is increased in critically ill
patients with sepsis and which proteolytic enzyme systems are involved in this degradation. Eight
patients and seven healthy volunteers were studied. In vivo muscle protein kinetics was measured
using arteriovenous balance techniques with stable isotope tracers. The activities of the major
proteolytic enzyme systems were analysed in combination with mRNA expression of genes related
to these proteolytic systems. Results show that critically ill patients with sepsis have a variable
but normal muscle PS rate, whereas protein degradation rates are dramatically increased (up
to 160 %). Of the major proteolytic enzyme systems both the proteasome and the lysosomal
systems had higher activities in the patients, whereas calpain and caspase activities were not
changed. Gene expression of several genes related to the proteasome system was increased in the
patients. mRNA levels of the two main lysosomal enzymes (cathepsin B and L) were not changed
but, conversely, genes related to calpain and caspase had a higher expression in the muscles of the
patients. In conclusion, the dramatic muscle wasting seen in critically ill patients with sepsis is due
to increased protein degradation. This is facilitated by increased activities of both the proteasome
and lysosomal proteolytic systems.

INTRODUCTION
Skeletal muscle wasting is a common feature in different
diseased states, e.g. cancer, sepsis, burns and diabetes,
but is seen in its worst form in patients treated in the
ICU (intensive care unit). Independent of their initial
diagnosis, most patients treated in the ICU for more than

24 h develop a syndrome called multiple organ failure in

which vital organ systems start failing and need to be
supported in the ICU for the patients to survive. Wasting
of muscle protein in these patients can be as much as
10 % per week in the initial weeks of ICU treatment [1]
and severely compromises muscle functionality both in
the ICU and long after discharge from the unit. Previous

Key words: calpain, caspase, critical illness, multiple organ dysfunction, muscle wasting, protein degradation, sepsis, skeletal muscle.
Abbreviations: AMC, 7-amido-4-methylcoumarin; Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-AMC; Ac-DEVD-CHO,
N-acetyl-Asp-Glu-Val-Asp aldehyde; Bnip3, Bcl2/adenovirus E1B 19 kDa interacting protein 3; COPD, chronic obstructive
pulmonary disease; DTT, dithiothreitol; FoxO3, forkhead box O3; ICU, intensive care unit; MuRF1, muscle RING-finger protein
1; NB, net balance; PB, protein breakdown; PS, protein synthesis; Ra , rate of appearance; Rd , rate of disappearance; SOFA, Sepsis
Organ Failure Assessment; SSA, 5-sulfosalisylic acid-2-dihydrate.
Correspondence: Dr Maria Klaude (email maria.klaude@ki.se).


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experiments from our group [2,3] indicate that protein

fractional synthesis rates in leg skeletal muscle on average
is not different in ICU patients compared with healthy
individuals but is far more variable. These observations
indicate that muscle wasting is mainly related to protein
degradation in this patient group.
In muscle cells, there are four main systems of
proteolytic enzyme pathways involved in protein
degradation: the ubiquitinproteasome pathway, the
autophagylysosome system, calpains and caspase 3.
Myofibrillar proteins are degraded by the ubiquitinproteasome pathway, and most cellular proteins degraded
by this pathway must first be polyubiquitinated in order
to be recognized by the 26S proteasome [4]. There are
three classes of enzymes involved in the initial steps of the
proteasome pathway: E1 proteins that activate ubiquitin,
E2 proteins that are ubiquitin-conjugating enzymes
and E3 protein ligases that transfer ubiquitin from the
conjugating system to the protein substrate [5]. Proteins
are then degraded in the 20S core of the 26S proteasome
by three different proteases into small peptides [6].
The autophagylysosome system is implicated in
the degradation of extracellular constituents, and the
turnover of cytoplasmic constituents and of cellular
organelles like mitochondria. Autophagosomes are
formed as double-membrane vesicles around cellular
constituents, and the vesicles fuse with lysosomes where
their contents are degraded. Cathepsins B, L, D and
H are the major proteolytic enzymes in lysosomes
and primarily determine the proteolytic capacity of this
organelle [7]. Calpains and caspase 3 are believed to be
involved in the initial step of myofibrillar breakdown by
releasing actin and myosin filaments from the myofibrils.
Skeletal muscle contractile proteins exist as actomyosin
complexes which cannot be degraded by the proteasome
but have to be released from the sarcomere as monomers
first [8].
The aim of the present study was to elucidate the
mechanisms leading to the dramatic muscle protein
loss in septic patients. To achieve this, we have used
various methods to measure PB (protein breakdown) in
ICU patients and healthy controls. We also measured
PS (protein synthesis) to confirm previous results
using a different method. Arteriovenous differences,
of phenylalanine and 3-methylhistidine tracers, over
leg muscles were used to quantify both PS and PB
in vivo. In vitro experiments of muscle biopsies were
used to analyse proteolytic activities of proteasomes,
lysosomes, calpains and caspase 3. In addition, mRNA
levels of components in the different pathways for
muscle catabolism were measured in the biopsies. This
is the first time that muscle protein kinetics and the
underlying mechanisms in the form of all proteolytic
enzyme activities and gene expression are measured in
the same group of actual critically ill patients with
sepsis.

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MATERIAL AND METHODS

Patients and subjects
ICU patients (n = 8) with sepsis and/or septic shock
at admittance were included in the study. Patients with
severe liver failure, undergoing dialysis or with impaired
coagulation not allowing muscle biopsies were excluded
from the study. The characteristics of the patients on
the study day are given in Table 1. According to the
SOFA (Sepsis Organ Failure Assessment) scores, all of
the patients suffered from multiple organ failure. All
of the patients were sedated, one with midazolam and
the others with propofol, together with intermittent
doses of analgesics, and all patients required mechanical
ventilation. At the time of the study, all patients were
circulatorily stabilized, although all but one patient
required vasopressor support. Intravenous short-term
corticosteroid treatment was given to seven patients
and one patient was given corticosteroid substitution.
Antibiotics were given to six patients and one patient
was treated with more than one. Antifungal therapy
was given to three patients. At the time of study one
of the patients had an indwelling thoracic epidural
catheter with continuous infusion of local anaesthetics
and short-acting opioids. Total parental nutrition, enteral
nutrition or a combination were given continuously
to all patients according to the individual caloric
goals (range 0.81.1 kcal/kg of body weight per h).
All patients received intravenous insulin to keep blood
glucose between 4 and 8 mmol/l. One patient died in
the ICU (day 40 of ICU stay). The rest survived ICU
and the hospital stay and were still alive 6 months after
completion of the study.
The control group consisted of eight healthy
volunteers; no data were available from one due to
technical problems with the tracer infusion. The patient
data were therefore compared with data of seven healthy
volunteers with a mean age of 25 years (range 2129)
consisting of five male and two female subjects with
normal BMI (body mass index).
The study protocol conformed to the ethical guidelines
of the 1975 Declaration of Helsinki and was approved
by the Ethical Committee of Karolinska Institutet,
Stockholm, Sweden. All subjects, or for the ICU patients,
close relatives, gave informed consent to participate in the
studies after receiving both oral and standardized written
information approved by the Ethical Committee.

Study protocol
The controls reported to the research laboratory in the
morning after an overnight fast (16 h). Patients were in
the ICU unit already for at least one day before inclusion
into the study.
Catheters were inserted in the radial artery and
in the cubital veins for the controls, whereas in the
patients existing arterial and central vein catheters were

Protein metabolism in human skeletal muscle

Table 1 Characteristics of patients on the study day

WBC, white blood count.

Diagnosis

Age

Gender

Days in ICU

SOFA

Body temperature ( C)

WBC (109 cells/l)

Six months survival

Cardiac arrest
Sepsis
COPD
Aspiration pneumonia
Respiratory failure
Tick-born encephalitis
Acute pancreatitis
Postoperative respiratory failure
Median values

51
66
61
68
59
69
55
76
63.5

Male
Female
Female
Male
Female
Male
Male
Female

18
7
5
2
3
5
6
1
5

12
12
7
9
10
12
13
10
11

39.8
38.1
37.0
36.2
38.0
38.5
38.6
38.4
38.3

11.9
36.8
9,4
20.5
11.1
15.4
13.4
9.4
12.7

No
Yes
Yes
Yes
Yes
Yes
Yes
Yes

used. Baseline blood samples were taken from the

artery. Subsequently, a primed continuous infusion of
[2 H5 ]phenylalanine (prime, 0.5 mg/kg of body weight;
infusion, 0.5 mg/kg of body weight per h) and 3[2 H3 ]methylhistidine (prime, 0.01 mg/kg of body weight;
infusion, 0.01 mg/kg of body weight per h) was started. At
the same time parenteral nutrition (Kabiven, Fresenius)
was started in the volunteers at 1 kcal/kg of body weight
per h. After 2 h, an additional catheter was placed in
the femoral vein. Blood samples from the femoral vein
and the radial artery were collected simultaneously at
220, 230 and 240 min after the start of the infusion.
Blood samples were centrifuged at 2000 g for 15 min to
obtain plasma which was stored at 80 C until analysis.
Immediately before and after the sampling period, blood
flow to the leg was measured by venous occlusion
plethysmography with a minimum of ten readings, as
described in detail previously [9]. Muscle biopsies were
taken at 220 and 240 min, immediately frozen in liquid
nitrogen and thereafter stored at 80 C. Muscle biopsies
were taken from the vastus lateralis using a Bergstrom
needle. The biopsies were taken using local anaesthesia
(lidocaine) and were confined to the skin and fascia only.

Amino acid analyses

Plasma samples were analysed for concentration
and tracer enrichment of phenylalanine- and 3methylhistidine by HPLC (Waters Alliance 2690 and
Waters fluorescence detector 474) and GCMS (Agilent
5973n) respectively.
For HPLC analysis, plasma was deproteinized with
3 % SSA (5-sulfosalisylic acid-2-dihydrate) containing
200 M norvaline as an internal standard. The HPLC
analysis has been described in detail previously [10].
Sample preparation and GCMS analysis to measure
plasma [2 H5 ]phenylalanine and 3-[2 H3 ]methylhistidine
have been described in detail previously [11].
Analyses of muscle tissue-free concentrations of
phenylalanine and 3-methylhistidine were performed
as described before [10]. For analyses of tissue-free
[2 H5 ]phenylalanine in muscle a modification of the

method used to analyse plasma [2 H5 ]phenylalanine

was utilized. The muscle sample was freeze-dried and
pulverized. Fat, connective tissue and blood were
carefully removed from the muscle fibres, which
were subsequently weighed again. Muscle samples were
homogenized in 6 % SSA using a mini-bead beater
(Biospec Products, Bartlesville, OK, U.S.A.). After
centrifugation, the amino acids in the supernatant
were purified using ion-exchange chromatography.
Furthermore, the samples were derivatized and analysed
on the GCMS as described for plasma samples [11]. The
samples were analysed five times and the median value
used for calculations to minimize the variation.

Enzyme measurements
Preparation of muscle biopsies
Approx. 90 mg of muscle tissue was homogenized in a
glass homogenizer in 1 ml of buffer A [50 mM Tris/HCl,
pH 7.5, 1 mM DTT (dithiothreitol), 1 mM EDTA,
5 mM MgCl2 , 250 mM sucrose and 10 % glycerol].
The homogenate was centrifuged at 700 g to remove
cell debris. The resulting supernatant was thereafter
centrifuged at 16 300 g. The supernatant was frozen
in aliquots at 80 C and used for determination of
proteasome and caspase 3 activity. The pellet was rinsed
in 1 ml of buffer B (0.15 M KCl and 20 mM sodium
phosphate, pH 6.0) and thereafter suspended in 150 l
of buffer B. The pellet suspension containing lysosomes
was frozen in aliquots at 80 C and used to determine
cathepsin activities. Before determination of protease
activities in the two fractions protein content was
measured using the BioRad protein assay. The pellet
suspension was first freezethawed three times.

Proteasome activity
Chymotrypsin-like activity of the proteasome fraction
was measured using the fluorogenic peptide substrate
SUC-LLVY-AMC [succinyl-Leu-Leu-Val-Tyr-AMC (7amido-4-methylcoumarin)] (Sigma) [12]. Then 10 l of
the supernatant (approximately 10 g of protein) was

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incubated in 100 l of buffer (50 mM Tris/HCl, pH 7.5,

1 mM ATP, 5 mM MgCl2 , 1 mM and 150 M LLVY)
in microplates. Standard curves were prepared using
AMC (Sigma). Fluorescence was measured continuously
over 1 h at 37 C in a FLOUstar OPTIMA (BMG
Labtechnologies) spectrofluorometer at ex = 380 nm and
em = 460 nm. Proteolytic activity was calculated from
the increment of the curves from samples and standards
and are expressed as pmol of AMC released/g of protein
per min.

1 mM DTT, 10 mM CaCl2 and 4.6 g/ml of BODIPY

FL-casein in a total volume of 220 l. Parallel incubations
were done in the absence of calcium and in the presence of
10 mM EDTA. Fluorescence was measured continuously
over 1 h at 26 C at ex = 485 nm and em = 520 nm.
Proteolytic activity was calculated from the increment
of the curves obtained and expressed as fluorescence
units/g of protein per min. By subtracting the activity
curve in the absence of calcium from the activity in the
presence of calcium, the calcium-dependent activity, i.e.
calpain activity, was obtained.

Cathepsin B and L activities

Activity of the lysosome fraction was measured using
Z-Arg-Arg-AMC (Sigma) for cathepsin B and Z-PheArg-AMC (Sigma) for cathepsin L [13]. For cathepsin B
activity, 10 l of pellet suspension (approximately 2 g
of protein) was incubated in 100 l of buffer (0.1 M
sodium phosphate, pH 6, 1 mM EDTA, 2 mM cysteine
and 250 M Arg-Arg-AMC). For cathepsin L activity,
10 l of pellet suspension (approximately 2 g of protein)
was incubated in 100 l of buffer (0.1 M sodium acetate,
pH 5.5, 1 mM EDTA, 1 mM DTT and 250 M PheArg-AMC). Standard curves were prepared using AMC.
Fluorescence and proteolytic activity was measured and
calculated as described for the proteasome activity.

Caspase 3 activity
Activity was measured using Ac-DEVD-AMC (Nacetyl-Asp-Glu-Val-Asp-AMC; Sigma) as substrate [14].
Aliquots of the supernatant (30 g of protein) were
incubated in 100 l of reaction buffer (100 mM Hepes,
pH 7.5, 10 % sucrose and 1 mM DTT) containing 90 M
DEVD-AMC at 30 C for 1 h. Parallel incubations were
done in the presence of 30 M Ac-DEVD-CHO (Nacetyl-Asp-Glu-Val-Asp aldehyde; Sigma) a caspase 3
inhibitor. Fluorescence and proteolytic activity were
measured and calculated as described for the proteasome
activity. Activity in the presence of Ac-DEVD-CHO
was subtracted from the activity in the presence of
Ac-DEVD-AMC alone and expressed as fluorescence
units/g of protein per min.

Calpain activity
Approx. 50 mg of muscle tissue was homogenized in a
buffer {20 mM Tris/HCl, pH 8, 5 mM EDTA, 10 mM
2-mercaptoethanol, 2.5 M E-64 [trans-epoxysuccinyll-leucylamido-(4-guanidino)butane], 2 mM PMSF and
0.1 mg/ml trypsin inhibitor}. The homogenate was
centrifuged 19 900 g for 30 min. Protein concentration
in the supernatant was determined as described above.
Calpain activity in the supernatant was determined using
BODIPY FL-casein (4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-propionic acid-labelled casein()
(Molecular Probes) as substrate [14]. Then, 30 g of
supernatant protein was incubated in a buffer containing
20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 100 mM KCl,

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Gene expression
Selected genes involved in the different proteolytic
enzyme systems were analysed using real-time PCR.
Total RNA was isolated from muscle samples using
TRIzol (Invitrogen) and quantified using a nano-drop
spectrophotometer. cDNA was prepared from 1 g of
RNA using random hexamer primers and reverse transcription reagents (Applied Biosystems) in a final volume
of 40 l. Oligonucleotide primers were designed using
a primer design centre (http://www.probelibrary.com/)
and synthesized by Invitrogen. To avoid amplification
of nuclear DNA the primers were designed to amplify
across exonexon boundaries. The cycle threshold
values (dCT ) were calculated based on correction to
GAPDH (glyceraldehyde-3-phosphate dehydrogenase)
and statistical analysis was applied to these raw data. The
linear value of each sample (2 dCt ) was calculated and this
value from each ICU patient was then compared with
the mean of all the control values to obtain an estimate
of the mean increase/decrease and the variation between
samples [15].

Calculations
Skeletal muscle protein kinetics over the leg was
performed using both a two- and a three-pool model. In
the two-pool model, the kinetics are estimated as the rate
of appearance (estimate for PB) and rate of disappearance
(estimate for PS), whereas in the three-pool model the
additional muscle measurements allows for calculations
of actual PB and synthesis rates.
The NB (net balance) of phenylalanine and 3methylhistidine (nmol/min per 100 ml of leg volume)
across the leg was calculated as:
NB = (CA CV ) F
where CA and CV are the arterial and venous
concentrations respectively (nmol/ml) and F is the plasma
flow (ml/min per 100 ml of leg volume).
The Ra (rate of appearance) and Rd (rate of
disappearance) of phenylalanine (nmol/min per 100 ml
of leg volume) across the leg were calculated utilizing the
two-pool model [11]:
Ra = CV [1 (EV /EA )] F

Protein metabolism in human skeletal muscle

Rd = NB + Ra
where EV and EA are enrichments of the plasma
phenylalanine tracer in the vein and artery respectively
(atom percent excess).
Leg skeletal muscle PS and PB (nmol/min per 100 ml
of leg volume) rates were calculated utilizing a three-pool
model for phenylalanine [16]:
PS = (CA EA CV EV ) F/EM
PB = PS NB
where EM is enrichment of the free phenylalanine tracer
in muscle (atom percentage excess). The mean enrichment
from the two biopsies was used for calculation.
The Ra of 3-methylhistidine over the leg was calculated
utilizing a two-pool model [11]:

Figure 1 NB, rates of PS and degradation in the leg

muscle of septic patients (n = 8) and controls (n = 7)
as determined by arteriovenous balance and phenylalanine
tracer

R d and R a were determined according to the two-pool model; PS and PB

were determined according to the three-pool model. Results are means + S.D.

P < 0.01 and P < 0.001 compared with the control value, as determined
using a Students t test.

Ra = CA [(EA /EV ) 1] F

Statistical methods
Students t tests were used to compare values between
ICU patients and controls. Results are means + S.D.

RESULTS
Muscle protein kinetics
The ICU patients had approx. 160 % higher muscle PB
compared with the controls as calculated from both
two-pool and three-pool models (Figure 1). The NB of
phenylalanine concentration across the leg was negative
in the patients, whereas the controls had a NB not
significantly different from zero (Figure 1). PS in leg
muscle did not differ between ICU patients and controls
as calculated from both two-pool Rd and three-pool
PS (Figure 1). In addition, the Ra of 3-methylhistidine
was significantly higher in the patients compared with
controls.

Figure 2 Proteasome proteolytic activity in muscle extracts from patients with sepsis (n = 8) and controls
(n = 7)

Results are means + S.D. P < 0.05 compared with the control value, as
determined using a Students t test.

Proteolytic activities
Proteasome activity was 44 % higher in the ICU patients
as compared with the controls (Figure 2). In addition
lysosomal proteolytic activities were higher in the
patients compared with the controls: cathepsin B by
200 % and cathepsin L by 150 % (Figure 3). In the ICU
patients, the proteasome activity correlated significantly
with both cathepsin B activity (R = 0.929, P < 0.001)
and cathepsin L activity (R = 0.860, P < 0.01), whereas
this correlation was not seen in the controls. The calpain
activity did not differ between patients and controls
(Figure 4). In addition, the caspase 3 activity did not
differ significantly between the patients and the controls
(Figure 4).

Figure 3 Proteolytic activities of cathepsin B and L in

muscle extracts from patients with sepsis (n = 8) and
controls (n = 7)

Results are means + S.D. P < 0.01 compared with the control values, as
determined using a Students t test.

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Figure 4 Proteolytic activities of calpain and caspase 3 in

muscle extracts from patients with sepsis (n = 8) and
controls (n = 7)

Results are means + S.D.

Correlations between in vitro and in vivo

measurements
In the patients all of the in vitro proteolytic activities
correlated significantly with the in vivo measurements
of Ra and PB using the phenylalanine tracer. Proteasome
activity correlated with Ra (R = 0.708, P < 0.05) and PB (R
= 0.783, P < 0.05). Cathepsin B correlated with Ra (R =
0.818, P < 0.02) and PB (R = 0.771, P < 0.05). Cathepsin
L correlated with Ra (R = 0.851, P < 0.01) and PB (R =
0.815, P < 0.02). In the controls proteasome activity
correlated significantly with Ra (R = 0.973, P < 0.001)
and PB (R = 0.962, P < 0.001), whereas no correlation
was found between cathepsin activities and the in vivo
measurements. Ra of 3-methylhistidine, however, did not
show any correlation with the in vitro measurements
of the proteolytic activities in either the patients or the
controls.

Gene expression
Real-time QPCR (quantitative PCR) was used to
determine whether genes related to different pathways for
skeletal muscle protein turnover were activated. We chose
genes related to the proteasome pathway, the autophagy
lysosome system, calpain and caspase (Figure 5). In
the ICU patients, there was a general tendency for
up-regulation of expression of the genes investigated
when compared with the controls. Expressions were
significantly higher in patients compared with controls
for atrogin-1 (by 200 %, P = 0.02), ubiquitin (by
200 %, P = 0.004), Bnip3 (Bcl2/adenovirus E1B 19 kDa
interacting protein 3) (by 800 %, P = 0.02), caspase
3 (by 300 %, P = 0.00002) and m-calpain (by 200 %,
P = 0.02) (Figure 5). Although cathepsin B and L
activities were significantly elevated in the patients
mRNA levels were not significantly different between
patients and controls. Conversely, calpain and caspase
3 mRNA levels were enhanced in the patients but the
enzyme activities remained at the same levels as in
the controls.

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Figure 5 Expression of genes related to the different

proteolytic pathways in muscle tissue

(A) Proteasome pathway, (B) autophagylysosome system, and (C) calpain and
caspase 3. The changes in mRNA levels of the patients (n = 8) are expressed
as a percentage of controls (n = 7) (horizontal line) and are means and
S.D. P < 0.05, P < 0.01 and P < 0.001 compared with the control
values (dC T ) as determined using a Students t test. GABARAPL1, GABAA
receptor-associated protein like 1; UBB, ubiquitin B; UBC, ubiquitin C.

DISCUSSION
The present study is the first to assess muscle wasting
in septic patients from in vivo kinetics measurements to
gene expression. In addition, these experiments were done
during continuous parenteral feeding, which is clinical
practice, in both the patients and the controls. The results
clearly show that in these critically ill patients the loss of
muscle mass is the result of increased protein degradation
rather than decreases in synthesis. It also shows that both
the proteasome and the lysosomal systems are activated
but that gene expression does not always support the
kinetic measurements.

Protein metabolism in human skeletal muscle

Protein kinetics
Earlier studies from our group have shown that
in vivo PS in skeletal muscle from patients with sepsis
does not differ from healthy controls when measured
as fractional synthesis rates using incorporation of
tracer phenylalanine [2,3]. In another study with similar
patients, we demonstrated an enhanced proteasome
activity in leg and respiratory muscles [12]. The present
study confirms that PS in skeletal muscle is unaltered
in septic patients showing that muscle wasting in these
patients is mainly due to increased protein degradation.
Here, we used an in vivo model measuring plasma flow
of tracer phenylalanine over the leg with three different
approaches (Figure 1). Ra and Rd were utilized to assess
protein kinetics in leg muscle using a two-pool model
[11]. The results showed that Ra was higher in the patients
compared with the controls, indicating an increase in PB,
whereas Rd indicated that PS was the same in both groups.
In the three-pool model, it is possible to calculate the PS
and PB rates of the leg muscle protein [16]. This model
also showed that compared with the controls the patients
had an increased PB and equal PS. The third model, a
measure of the Ra of 3-methylhistidine that is a marker of
contractile PB rates, showed an enhanced efflux rate in the
patients compared with the controls. These differences
are not the result of the age difference between our control
subjects and the patients, as human aging is characterized
by a decreased or maintained muscle PS and, if anything,
a decreased muscle PB [17,18].
In the present study, we also investigated the
proteolytic activities in all four main degradation
pathways in leg muscle biopsies: proteasome activity,
activities of lysosomal cathepsins B and L, and of calpain
and caspase 3. In addition, we measured mRNA levels of
proteins in respective pathways. We think it is important
to measure both protein activity and gene expression in
the same samples, as protein levels or activities are not
always regulated on the transcriptional but also on the
post-transcriptional level.

Ubiquitinproteasome pathway
There are a number of studies, using animal models for
sepsis, which have shown that in skeletal muscle the
ubiquitinproteasome pathway is up-regulated during
sepsis, both at the mRNA level of different components in
the pathway and proteolytic activities [1924]. However,
studies of molecular mechanisms of muscle atrophy
in human sepsis are few and the data from these
studies are not as comprehensive as the data from
animal experiments. In accordance with the animal
models for sepsis, elevated expression of ubiquitin in
skeletal muscle from septic patients was found [2528].
Furthermore, an induction of atrogin-1 [25], increased
mRNA levels of cathepsin B [26] and the proteasome
subunit HC3 [28] and enhanced proteolytic activity of
the proteasome [12,29] have been demonstrated in septic

patients. Muscle biopsies from septic patients in the

present study showed increased proteolytic activity of
the proteasome as well as enhanced mRNA levels
of factors linked to the induction of the ubiquitin
proteasome pathway (ubiquitin and atrogin-1, but not
MuRF1), thus indicating an enhanced capacity for PB
(Figures 2 and 5). Atrogin-1 and MuRF1 (muscle RINGfinger protein 1) are ubiquitin-ligases and increased
mRNA levels have been observed in a number of
animal models of muscle atrophy, including burn injury,
diabetes mellitus, denervation, unweighting and sepsis
[30]. However, studies of these two so-called atrogins in
human inflammatory states are few and there have been
inconsistent findings of mRNA levels of the two genes.
In COPD (chronic obstructive pulmonary disease) both
an elevation and no significant change of atrogin-1 and
MuRF1 gene expressions have been reported [31,32].

Autophagylysosome pathway
Recent data from animal experiments in vitro and in vivo
have indicated that the autophagylysosome system is
also involved in muscle atrophy and that this system
is co-ordinated with the ubiquitinproteasome system.
Furthermore, the induction of the two degradation
systems is mediated by transcription factor FoxO3
(forkhead box O3) [33,34]. In addition, results from a
human study on ventilator-induced diaphragm disuse
in patients after cerebrovascular accidents point in this
direction [35]. Earlier studies of models for sepsis in rats
have measured enhanced levels in muscle of cathepsin B
activity [36,37] and an elevation of cathepsin L mRNA
and protein levels [38]. There are also a few human
studies indicating an up-regulation of the lysosomal
system in skeletal muscle in catabolic states. In trauma
patients, increased levels of cathepsin D mRNA were
found [39] and increased levels of cathepsin B mRNA
and enzymatic activity were observed [40]. Cathepsin
B mRNA levels were increased in skeletal muscle of
patients with early lung cancer [41]. In patients with sepsis
immunolabelling of cathepsin B was enhanced in atrophic
muscle fibres [26]. In our present study, we found a
dramatic increase in activity of lysosomal cathepsin B
and L in leg muscle of the septic patients as compared
with the controls (Figure 3). However, there was no
difference in the mRNA levels of the cathepsins between
patients and controls (Figure 5). Studies using animal
models of muscle wasting also found limited effects on
mRNA levels of cathepsins but large increases in the
enzymatic activities, suggesting that post-transcriptional
processes may be mainly responsible for the activation
of cathepsins [4244]. In accordance with the animal
and human studies mentioned above [33,35] we found
increased levels of Bnip3 mRNA in the patients (Figure 5).
Bnip3 is a factor that recruits the autophagy machinery
on mitochondria [45]. Although both cathepsin B and L
are capable of degrading several myofibrillar proteins [7],

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skeletal muscle contains few lysosomes in comparison

with other organs such as the liver or spleen. Even if not
ruled out, it is unlikely that these organelles are involved
in the turnover of myofibrillar proteins. Mitochondria are
turned over via the autophagylysosome system and
are a better candidate for degradation after the activation
of this system [46]. Previously, we found a mitochondrial
content that was 3040 % lower in skeletal muscle of ICU
patients with sepsis and that this was not the result of
decreased biogenesis [15,47]. The co-ordinated induction
of the two main proteolytic pathways presumably
leaves the mitochondrial to myofibrillar composition
ratio relatively normal but with reduced strength and
endurance capacity due to the loss of myofibrillar
components and mitochondria [46]. In addition, our
results also indicate that the proteasome and autophagy
lysosome systems are activated simultaneously since
there was a significant positive correlation between
proteasome and lysosomal cathepsin activities in the
patients but not in the controls, suggesting a common
control system such as FoxO3.

Calpain and caspase systems

There is increasing evidence that calpain and/or caspase
3 are involved in the initial degradation of myofibrillar
proteins [8,48]. Since myofibrils are too large to be
engulfed by proteasomes, myofilaments must first
be released from the myofibrils prior to degradation
by the proteasome system. In contrast to the results of
cathepsins, the ICU patients in the present study had
elevated levels of mRNA of both m-calpain and caspase
3 but the activities of the two proteases did not differ
between the study groups (Figures 4 and 5). The question
is whether this discrepancy of activity compared with
expression levels depends on methodological difficulties,
due to low enzyme activities in the muscle extracts,
or reflects a high turnover of the enzymes. Animal
models for sepsis have come to different conclusions.
In septic rats increased mRNA levels of m- and calpain were found but no change in calpain activity
[49]. Wei et al. [14] found increased calpain activity in
septic rats but unchanged levels of caspase 3 mRNA
and enzyme activity. In two other studies of rodent
models for sepsis, increased levels of both enzyme
activity and protein levels of caspase 3 and calpain were
observed [48,50]. So far there have been no studies
carried out on calpain and caspase 3 in human sepsis
but a study of patients with acute quadriplegic myopathy
found enhanced immunoreactivity for calpain in atrophic
muscle fibres [51].

General conclusions
In the present study, we show that the dramatic loss
of skeletal muscle in critically ill patients with sepsisinduced multiple organ failure is due to an increased
protein degradation rather than a decreased PS. We also

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see that the patients continuously loose muscle protein

despite being fed, in contrast with the controls that did not
lose muscle protein but were in net protein balance. This
increased protein degradation is facilitated by increased
activities of the proteasome and lysosomal systems but
not of calpain and caspase. The results also show that
measuring mRNA levels will give indications of preor post-translational adaptations but are not alternative
measurements for enzyme activity or kinetic analyses.

AUTHOR CONTRIBUTION
Maria Klaude performed the experiments and data
analyses of the proteolytic enzymes, and participated in
the study design. Maiko Mori participated in the clinical
studies, performed the experiments and data analyses
of the protein kinetics, and participated in the study
design. Inga Tjader performed the clinical studies and
compiled the patient data. Thomas Gustafsson performed
the experiments and data analyses of gene expression.
Jan Wernerman participated in study design and clinical
studies. Olav Rooyackers participated in the study
design and supervised the clinical studies. Maria Klaude
compiled the data and wrote the paper. Maiko Mori, Inga
Tjader, Thomas Gustafsson, Jan Wernerman and Olav
Rooyackers all reviewed and revised the paper.

ACKNOWLEDGEMENT
We thank Ms Viveka Gustafsson for excellent nursing
assistance.

FUNDING
This work was supported by the Swedish Medical
Research Council [grant numbers 04210 and 14244].

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Received 9 May 2011/25 August 2011; accepted 31 August 2011

Published as Immediate Publication 31 August 2011, doi:10.1042/CS20110233


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